The use of organic insecticides is under significant public scrutiny because of the environmental problems they cause and the high risks their use poses to human health. Thus there is considerable interest in the development of alternate strategies for insect control. One approach that has received significant interest in recent times is the development of Bacillus thuringiensis toxins as insecticides. These bacterial derived products have, however, met with only limited success in pest control because they are too specific in their host range, and usually only moderately efficacious. Attempts to improve their efficacy have failed to date because little effort has been devoted to understanding that molecular basis for the specificity and insecticidal properties of these toxins. Thus the long-term objective of the project is to understand the mode of action of the toxins of the bacterium, B. t. israelensis. The focus of the current on-going grant is principally on the 25kDa cytolytic toxin, a tryptic fragment of the 27kDa toxin. The principal focus of the proposal is to characterize the mechanism of membrane binding of the 25kDa toxin of B. t. israelensis. This objective will be achieved by utilizing monoclonal antibody probes that have been developed against the binding domains of the toxin. Attempts will also be made to determine the structure of these domains. Other aspects of the research program involve attempts to characterize the mode of action of the mosquitocidal 72kDa toxin. Characterization of the mechanism of action will be achieved with a resistant mosquito line that has been developed, by evaluating how the toxin interacts with midgut toxin receptors, and also by utilizing monoclonal antibodies to the toxin as probes. Finally attempts will be made to isolate the toxin receptor from the mosquito midgut using a variety of biochemical and immunological approaches. The characterization of the B. thuringiensis toxin receptors will facilitate the rational design and use of B. thuringiensis toxins for mosquito control and thereby significantly lowering the risk of resistance development in these pest species which are of major concern in human health. This work will contribute substantially to our understanding of the action of toxins that are likely to see increased use in mosquito control programs. In addition, the characterization of the toxin receptors will also provide critical information on how mosquitoes regulate ion concentrations in the midgut. Furthermore as it has been hypothesized that B. thuringiensis toxins potentially affect the amino acid/K+ symport in the midgut columnar cell apical membranes the characterization of B. T. israelensis toxin receptors would also provide fundamental knowledge for investigators interested in ion regulation in insects.